The detection and analytical confirmation of synthetic fentanyl analogues in human urine and serum using an Agilent Ultivo LC/TQ

Significance of the Topic

This study addresses the growing challenge of detecting and confirming illicit synthetic fentanyl analogues and related opioids in biological fluids. Rapid emergence of novel analogues demands analytical methods that deliver high sensitivity, precision, and throughput for clinical, forensic, and research laboratories.

Objectives and Study Overview

The primary goal was to develop and validate a targeted LC–MS/MS method on the Agilent Ultivo triple quadrupole platform to quantify 12 fentanyl analogues, the precursor 4-ANPP, and the potent opioid mimic W-18 in human serum and urine. The study compared analytical performance across both matrices, evaluating limits of quantitation, linearity, accuracy, and precision.

Methodology

Sample preparation involved protein precipitation of 250 µL serum or urine aliquots with cold acetonitrile containing deuterated internal standards, followed by centrifugation and dilution. Calibration curves spanned 1 pg/mL to 500 ng/mL, employing matrix-matched standards. Data acquisition and quantitation used Agilent MassHunter software and optimized MRM transitions.

Used Instrumentation

• Agilent 1290 Infinity II LC system (pump, autosampler with thermostat, multicolumn thermostat)
• Agilent Poroshell 120 EC-C18, 2.1×50 mm, 2.7 µm column at 55 °C
• Mobile phases: 0.01 % formic acid/5 mM ammonium formate in water (A), 0.01 % formic acid in methanol (B)
• Flow rate: 0.4 mL/min, 7 min gradient cycle
• Agilent Ultivo Triple Quadrupole MS with ESI in positive MRM mode

Main Results and Discussion

The method achieved excellent linearity (R2 > 0.996) over 10–500 ng/mL in serum and 50–500 ng/mL in urine. Lower limits of quantitation ranged from 10 pg/mL to 100 pg/mL, with interbatch precision (%RSD) below 9 % across all levels. Representative MRM chromatograms confirmed clear separation and consistent response for all analytes.

Benefits and Practical Applications

This protocol enables simultaneous quantitation of multiple fentanyl analogues with high throughput and minimal sample preparation. It supports forensic toxicology, clinical pharmacology, and quality control by delivering rapid, reliable data on emerging synthetic opioids.

Future Trends and Applications

Ongoing work should focus on expanding the panel to new analogues as they appear, refining sample cleanup to mitigate matrix effects, and integrating automated data review for real-time monitoring in clinical and forensic settings.

Conclusion

The validated Agilent Ultivo LC/TQ method offers robust, sensitive, and precise quantitation of a broad spectrum of synthetic fentanyl opioids in serum and urine. Its performance characteristics make it a powerful tool for laboratories confronting the dynamic landscape of illicit opioid detection.